إعداد المانحة والحصاد القلب : <ol style=";text-align:right;direction:rtl"><li style="text-align: left;text-align:right;direction:rtl"> الفأر هو تخدير مع حقن داخل الصفاق من مستلق بنتوباربيتال ووضعها على ارض الملعب من المنطوق. </li><li style="text-align: left;text-align:right;direction:rtl"> يتم إجراء شق البطن طويلة خط الوسط. وتراجع محتويات البطن بشاش إلى اليسار ، من أجل كشف الشريان الأورطي البطني والوريد الأجوف السفلي. </li><li style="text-align: left;text-align:right;direction:rtl"> يتم حقن 1 مل من الهيبارين (10u/ml) في الوريد الأجوف السفلي للheparinization. </li><li style="text-align: left;text-align:right;direction:rtl"> يتم إجراء بضع الصدر : يتم تقسيم الضلوع على جانبي العمود الفقري والصدر الجدار الأمامي هو levered تصل إلى فضح القلب. </li><li style="text-align: left;text-align:right;direction:rtl"> و تربط الوريد الأجوف السفلي ، والوريد الأجوف العلوي والوريد مع المفردة 6-0 الحرير وتنقسم متفوقة على الحروف المركبة. </li><li style="text-align: left;text-align:right;direction:rtl"> يتم فصل الشريان الأورطي والشريان الرئوي تصاعدي ومقسمة على النحو بشكل أقصى قدر ممكن. </li><li style="text-align: left;text-align:right;direction:rtl"> و تربط الأوردة الرئوية القاصي ومقسمة إلى العلاقات بين البلدين. </li><li style="text-align: left;text-align:right;direction:rtl"> هو perfused القلب مع 4 درجة مئوية حل المالحة. </li><li style="text-align: left;text-align:right;direction:rtl"> يتم تخزين الجليد في القلب في حل المالحة الباردة. </li></ol> إعداد المتلقي وزرع <ol style=";text-align:right;direction:rtl"><li style="text-align: left;text-align:right;direction:rtl"> الفأر هو تخدير مع حقن داخل الصفاق من مستلق بنتوباربيتال ووضعها على ارض الملعب من المنطوق. </li><li style="text-align: left;text-align:right;direction:rtl"> يتم إجراء شق البطن طويلة خط الوسط. وتراجع محتويات البطن خارج البطن بشاش لفضح الشريان الأورطي البطني والوريد الأجوف السفلي. </li><li style="text-align: left;text-align:right;direction:rtl"> يتعرض لها فروع الشريان الأورطي البطني والوريد الأجوف السفلي وligated مع 10-0 الغرز. </li><li style="text-align: left;text-align:right;direction:rtl"> توضع الأربطة الداني والقاصي حول الأبهر والوريد الأجوف السفلي على التوالي. </li><li style="text-align: left;text-align:right;direction:rtl"> يتم إجراء فصد في الوريد الأجوف السفلي من المتلقي مع إبرة قياس 30. ثم تم تمديده لافتتاح بطول يساوي الأبهر والرئوي مع المانحين الدقيقة المقص. </li><li style="text-align: left;text-align:right;direction:rtl"> يتم إجراء بضع الأبهر في الشريان الأورطي البطني للمتلقي مع إبرة قياس 30. </li><li style="text-align: left;text-align:right;direction:rtl"> والمروية الأورطى فتح البطن والوريد الأجوف السفلي بمحلول ملحي. </li><li style="text-align: left;text-align:right;direction:rtl"> يوضع القلب المتبرع على الجانب الأيمن من البطن المستلم وتتناول بشاش. </li><li style="text-align: left;text-align:right;direction:rtl"> يتم وضع الغرز البقاء في المؤسسات الرئيسية القريبة والبعيدة من الشريان الأورطي البطني المتلقي مع الجهة المانحة الأبهر الصاعد. </li><li style="text-align: left;text-align:right;direction:rtl"> الانتهاء من مفاغرة من الجانب الأيسر من الشريان الأورطي البطني المتلقي والمتبرع الأبهر الصاعد مع استمرار الغرز. </li><li style="text-align: left;text-align:right;direction:rtl"> تصنع الخيوط البقاء الأولين في ذروة الدانية والقاصية من الوريد قمة المستلم الأجوف السفلي مع الشريان الرئوي الجهة المانحة للمفاغرة من الشريان الرئوي المتبرع والمتلقي في الوريد الأجوف السفلي. </li><li style="text-align: left;text-align:right;direction:rtl"> إغلاق الجدار الأيسر من الوريد الأجوف السفلي والشريان الرئوي مع المانحين الغرز المستمرة في داخل الوريد الأجوف السفلي. </li><li style="text-align: left;text-align:right;direction:rtl"> بعد واحد عقدة مع خياطة قمة البقاء الداني ، يتم إغلاق الجدار الأيمن من الوريد الأجوف السفلي والشريان الرئوي الجهة المانحة مع استمرار الغرز خارج الكهف الوريد السفلي. </li><li style="text-align: left;text-align:right;direction:rtl"> يتم تشغيل القلب المتبرع عبر إلى الجانب الأيمن من البطن المستلم. غلقت جدار الشريان الأورطي حق المتلقي في البطن والجهة المانحة مفاغرة الأبهر الصاعد مع استمرار الغرز. </li><li style="text-align: left;text-align:right;direction:rtl"> بعد إجراء مفاغرة ، تتم إزالة ربطة قاصية للتحقق من نزيف من مفاغرة. </li><li style="text-align: left;text-align:right;direction:rtl"> إذا كان هناك القليل لعدم وجود نزيف من مفاغرة ، تتم إزالة ربطة القريبة. قلب يملأ المتبرع بالدم فورا ، ولون القلب تصبح حمراء وتبدأ مرة أخرى للفوز. </li><li style="text-align: left;text-align:right;direction:rtl"> يتم إرجاع الأمعاء إلى البطن ، والتي هي مغلقة مع الغرز 6-0 المستمر. </li><li style="text-align: left;text-align:right;direction:rtl"> يتم وضع الماوس المتلقي على منطقة دافئة. بعد ساعة واحدة المتلقي للشفاء. </li></ol>

<ol>
	<li>Corry, R. o. b. e. r. t. . J., Winn, H. e. n. r. y. . J., Russell, P. a. u. l. . S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primarily+vascularized+allografts+of+heart+in+mice.">Primarily vascularized allografts of heart in mice.</a> Transplantation. 16, 343-350 (1973).</li></ol>

النتائج لقد أجرينا بنجاح أكثر من 1000 حالة لزرع القلب في الفئران منتبذ وحقق أكثر من 90 ٪ نسبة النجاح في سلالات مختلفة من الفئران ، بما في ذلك نوع البرية ، الفئران المعدلة وراثيا والجينات خروج المغلوب. مناقشة منذ الدكاترة. وقدم كوري راسيل منتبذ زرع القلب في الفئران في عام 1973 ، أثبت هذا النموذج لتكون ذات قيمة خاصة لدراسة استجابة مناعية الرفض ووضع استراتيجيات للمناعة الرواية. ويمكن استخدام هذا النموذج على نطاق أوسع الآن بسبب ظهور العديد من الفئران المعدلة وراثيا بالضربة القاضية جين قدمت طرقا جديدة لدراسة آليات الرفض / التسامح. قد زرع قلب أوعية دموية تكون نموذجا أكثر ملاءمة لعمليات زرع الأعضاء الصلبة السريرية. على الرغم من أن عمليات زرع الجلد أو تحت الجلد لزرع قلوب الأطفال حديثي الولادة هي أبسط من الناحية التقنية ، واتساع الأوعية الدموية ما هو مطلوب للبقاء الكسب غير المشروع في هذه زرع حساسة بشكل خاص لردود خيفي. وهكذا ، والجلد وتحت الجلد سماع زرع يمكن أن تكون مفيدة لاختبار صارم للتسامح ، ولكن ليس من أجل تطوير أو تحسين بروتوكولات إحداث مناعة أو التسامح. تعديل نحن زرع القلب الوعائية التقنية في عدة طرق لتحسين الكفاءة ومعدلات النجاح : <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وينبغي أن تروى القلب المتبرع بمحلول ملحي من الأبهر الصاعد حتى يصبح لون القلب الخفيفة بعد الحصاد. </li><li style=";text-align:right;direction:rtl"> وينبغي ligated فروع الشريان الأورطي البطني والوريد الأجوف السفلي من المتلقي مع 10-0 خياطة الجروح ، ومنع النزيف أثناء عملية التجلط وبعد العملية. </li><li style=";text-align:right;direction:rtl"> وينبغي أن تروى تجويف الشريان الأورطي البطني والوريد الأجوف السفلي من المتلقي مع المحلول الملحي حتى لا يترك الدم. هذا يمكن منع تجلط الدم بعد العملية. </li><li style=";text-align:right;direction:rtl"> وينبغي تجنب فقاعات الهواء داخل الأوعية عند اتخاذ anastomoses. </li><li style=";text-align:right;direction:rtl"> ينبغي أن anastomoses الشريان 11-0 استخدام الغرز ، للمساعدة في منع النزف من anastomoses. </li><li style=";text-align:right;direction:rtl"> يجب على شق من الشريان الأورطي البطني والوريد الأجوف السفلي للمتلقي يكون مساويا لفتح الشريان الرئوي والشريان الأبهر الصاعد من القلب المتبرع. </li><li style=";text-align:right;direction:rtl"> فمن المهم أن يكون هناك مجال تشغيل واضحة وتوسعية ، وبالتالي استخدام 6-0 خياطة بدلا من المشابك لانسداد الشريان الأورطي مؤقتا والوريد الأجوف السفلي. </li></ol> استنتاج على الرغم من أن تقنية زرع القلب في الفئران منتبذ يمثل تحديا تقنيا ، وهو أسلوب مهم لدراسة alloresponses. وقد أدت الممارسات والتقنية تعديلات في معدل نجاح اكثر من 90 ٪ في المختبر لدينا. ونحن نعتقد أن الفيديو والمخطوطة المرفقة سوف تساعد على تقليل منحنى التعلم بالنسبة للمختبرات التي ترغب في استخدام هذه التقنية.

heterotopic heart transplantation in mice

وقد استخدمت في عمليات زرع القلب منتبذ الماوس على نطاق واسع منذ أن تم عرضه من قبل الدكاترة. كوري و راسل في عام 1973. هو قيمة خاصة لدراسة ورفض الاستجابة المناعية الآن أن أجدد المعدلة وراثيا والفئران بالضربة القاضية جين المتاحة ، ولقد تم وضع عدد كبير من الكواشف المناعية. نموذج زرع القلب هو أقل صرامة من النماذج زرع الجلد ، وعلى الرغم من أن أكثر تحديا من الناحية التقنية. قمنا بتطوير تقنية تعديل وأكملت أكثر من 1000 حالة ناجحة لزرع قلب منتبذ في الفئران. عند اتخاذ مفاغرة من الأبهر الصاعد والأبهر البطني ، يتم وضع الغرز بقاء اثنين في المؤسسات الرئيسية القريبة والبعيدة من الشريان الأورطي البطني المتلقي مع الجهة المانحة ليالي الأبهر الصاعد ، ثم باستخدام 11-0 خياطة لمفاغرة على جانبي الأبهر مع استمرار الغرز. الغرز البقاء جعل مفاغرة أسهل و11-0 هو حجم خياطة المثالي لتجنب النزيف والتجلط. عند اتخاذ مفاغرة من الشريان الرئوي والوريد الأجوف السفلي ، يتم إجراء الغرز اثنين في البقاء على قمة الدانية والقاصية من الوريد قمة المتلقي ليالي الأجوف السفلي مع الشريان الرئوي المانحة ق. إغلاق الجدار الأيسر من الوريد الأجوف السفلي والشريان الرئوي مع الجهات المانحة ليالي الخيوط المستمرة في داخل الوريد الأجوف السفلي بعد ، واحد مع بقاء عقدة قمة القريبة خياطة الجدار الأيمن من الوريد الأجوف السفلي والشريان الرئوي الجهة المانحة ليالي هي مغلقة مع استمرار الغرز خارج الكهف الوريد السفلي مع 10-0 الغرز. هذا الأسلوب الأسهل لتنفيذ لأن يتم مفاغرة فقط على جانب واحد من الوريد الأجوف السفلي والغرز 10-0 هو الحجم المناسب لتجنب النزيف والتجلط. في هذه المقالة ، ونحن نقدم تفاصيل هذه التقنية لاستكمال الفيديو.

Watch this Scientific Journal Video about Heterotopic Heart Transplantation in Mice at JoVE.com

Heterotopic Heart Transplantation in Mice

وقد ثبت الماوس زرع قلب منتبذ النموذج من قبل محققين كثير أن يكون وسيلة هامة لدراسة آليات الرفض والاستجابة المناعية. ومع ذلك ، فإن التقنيات المستخدمة لا تزال صعبة. عن طريق تعديل معيار تقنيات لقد حققنا نجاحا مع أكثر من 1000 عملية زرع.

منتبذ زرع القلب في الفئران

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable ...

heterotopic-heart-transplantation-in-mice

Universidad San Sebastian

Research

JoVE Journal

Biology

29.4K Views.  University of California, San Francisco - UCSF. وقد ثبت الماوس زرع قلب منتبذ النموذج من قبل محققين كثير أن يكون وسيلة هامة لدراسة آليات الرفض والاستجابة المناعية. ومع ذلك ، فإن التقنيات المستخدمة لا تزال صعبة. عن طريق تعديل معيار تقنيات لقد حققنا نجاحا مع أكثر من 1000 عملية زرع.

Video: Heterotopic Heart Transplantation in Mice

Small Bowel Transplantation In Mice

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive medications, infection prophylaxis, and suitable patient selection helped improve actuarial graft and patient survival rates for all types of intestine transplantation. Patients with irreversible intestinal failure and complications of parenteral nutrition should now be routinely considered for small intestine transplantation. However, Survival rates for small intestinal transplantation have been slow to improve compares increasingly favorably with renal, liver, heart and lung. The small bowel transplantation is still unsatisfactory compared with other organs. Further progress may depend on better understanding of immunology and physiology of the graft and can be greatly facilitated by animal models. A wider use of mouse small bowel transplantation model is needed in the study of immunology and physiology of the transplantation gut as well as efficient methods in diagnosing early rejection. However, this model is limited to use because the techniques involved is an extremely technically challenging. We have developed a modified technique. When making anastomosis of portal vein and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s portal vein. The left wall of the inferior vena cava and donor s portal vein is closed with continuing sutures in the inside of the inferior vena cava after, after one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s portal vein are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis.  In this article, we provide details of the technique to supplement the video.

The mouse small bowel transplantation model has been recognized as an important tool to study mechanismes of immune rejection and screen new immunosuppressive drugs. However, this model is limited to use because the techniques involved is an extremely technically challenge. Now we introduce the modified technique.

زرع الأمعاء الصغيرة في الفئران

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive ...

Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice. We found that it was easier to concentrate the islets or cells into pellets in the final delivery tubing (PE50) used to transplant the cells under the kidney capsule. This technique provides both speed and ease while reducing any undue stress to the cells or to the mouse.

Loading: Settled, hand picked, islets or pelleted cells are carefully aspirated off the bottom of a 1.5 mL microcentrifuge tube using a p200 pipetteman and a straight, thin-wall pipette tip. A length of PE50 tubing is attached to the pipette tip using a small silicone adapter tubing. Cells are allowed to settle, in the tip, and then are transferred to the PE50 tubing by slowly dialing the pipetteman. Once the cells are near the end of the PE50 tubing, a kink is made and the silicone adaptor tubing is placed over the kink. The PE50 tubing is transferred to a 15 mL conical containing a cut 5 mL pipet, and the PE50 tubing is taped over the side of the 5 mL pipet to prevent curling during centrifuging. Cells are allowed to reach 1,000 rpm and stopped.
Transplantation: Recipient mice are anesthetized, shaved, and cleaned. A small incision is made on the left flank of the mouse and the kidney is exposed. The kidney, fat, and tissue are kept moist with normal saline swab. The distal end of the PE50 is attached to a Hamilton screw drive syringe, containing a pipette tip, using the silicone adaptor tubing. A small nick is made on the right flank side of the kidney, not too large nor too deep. The beveled end of the PE50 tubing, nearest the cells, is carefully placed under the capsule, the tubing is moved around gently to make space while swabbing normal saline; a dry capsule can tear easily. A small air bubble is delivered under the capsule by slowly dialing the syringe screw drive. Islets are then slowly delivered behind the air bubble. Once the islets have been delivered kidney homeostasis is maintained and the knick is cauterized with low heat. The kidney is placed back into the cavity and the peritoneum and skin are sutured and stapled. Mice are immediately treated with Flunixin and Buprenorphine s.q. and placed in a cage on a heating pad.

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of mice. Cells are concentrated into pellets in the final tubing used for transplanting the cells under the kidney capsule. The ease of this technique reduces stress to the cells and the mouse.

زرع البنكرياس من الجزر في الكبسولة الكلى من الفئران من السكري

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice.  We found that it was easier ...

Murine Skin Transplantation

As one of the most stringent and least technically challenging models, skin transplantation is a standard method to assay host T cell responses to MHC-disparate donor antigens. The aim of this video-article is to provide the viewer with a step-by-step visual demonstration of skin transplantation using the mouse model. The protocol is divided into 5 main components: 1) harvesting donor skin; 2) preparing recipient for transplant; 3) skin transplant; 4) bandage removal and monitoring graft rejection; 5) helpful hints. Once proficient, the procedure itself should take &lt;10 min to perform.

Allogeneic skin transplantation is a standard model to assay host T cell responses to MHC-disparate donor antigens. This video-article provides a visual tutorial of each step involved in performing a BALB/c--&gt;C57BL/6 skin transplant.

زرع الجلد الفئران

As one of the most stringent and least technically challenging models, skin transplantation is a standard method to assay host T cell responses to ...

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

جيل من الخلايا البشرية باء تنشيط CD40

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Visualizing the Beating Heart in Drosophila

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in myogenic hearts. A key step in examining heart function in the fly is finding a way to access the heart in a manner that preserves its myogenic function while still allowing the beating heart organ to be observed and recorded. Two different methods for observing and recording the beating heart in both larva and adult Drosophila are described here. Our semi-intact preparation using adult flies allows clear visualization of the abdominal heart without interference from the pigmented cuticle and overlying fat bodies. To record larval heart beats it is necessary to immobilize the larva, which minimizes body wall movements thereby reducing heart movements that are not associated with myocardial contractions. Our methodologies produce stable adult and larval heart preparations that can beat for hours at rates of 1-3 Hz.

Visualizing the Beating Heart in Drosophila

Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology.

تصور القلب النابض في ذبابة الفاكهة</em

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

Heterotopic and Orthotopic Tracheal Transplantation in Mice used as Models to Study the Development of  Obliterative Airway Disease

Obliterative airway disease (OAD) is the major complication after lung transplantations that limits long term survival (1-7).
To study the pathophysiology, treatment and prevention of OAD, different animal models of tracheal transplantation in rodents have been developed (1-7). Here, we use two established models of trachea transplantation, the heterotopic and orthotopic model and demonstrate their advantages and limitations.
For the heterotopic model, the donor trachea is wrapped into the greater omentum of the recipient, whereas the donor trachea is anastomosed by end-to-end anastomosis in the orthotopic model.
In both models, the development of obliterative lesions histological similar to clinical OAD has been demonstrated (1-7).
This video shows how to perform both, the heterotopic as well as the orthotopic tracheal transplantation technique in mice, and compares the time course of OAD development in both models using histology.

This video shows and compares two experimental models to study the development of obliterative airway disease (OAD) in mice, the heterotopic and orthotopic tracheal transplantation model.

زرع القصبة الهوائية ومنتبذ مثلي في الفئران المستخدمة كنماذج لدراسة الأمراض تنمية إيرواي مسد

Obliterative airway disease (OAD) is the major complication after lung transplantations that limits long term survival (1-7).
To study the ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

إعداد الأجنة المجهري للإلكترون من ذبابة الفاكهة أنبوب القلب الجنينية

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

الكمي بؤر γH2AX في الاستجابة للإشعاع المؤين

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Evaluation of the Spatial Distribution of &gamma;H2AX following Ionizing Radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as &gamma;H2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of &gamma;H2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds4.
Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of &gamma;H2AX as molecular marker of DSBs, a disparity in &gamma;-irradiation-induced &gamma;H2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of &gamma;H2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between &gamma;H2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of &gamma;H2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.

Evaluation of the Spatial Distribution of &#947;H2AX following Ionizing Radiation

Microscopic analysis of &gamma;H2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced &gamma;H2AX formation within the nucleus.

تقييم التوزيع المكاني للγH2AX التالية الإشعاعات المؤينة

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

مولد الرمع الفحص : خلايا منتسب

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...